The immature erythrocytes in blood are called reticulocytes and the reticulocyte count is considered to be a laboratory parameter of great significance in the diagnosis of acute internal hemorrhage and hemolytic anemia, among other diseases. This is because reticulocytes serve as an indicator of blood cell production (erythrocyte differentiation).
It is also known that the reticulocyte has RNA and in the determination of reticulocytes, it is common practice to stain a blood smear with new methylene blue (NMB), brilliant cresyl blue (BCB) or the like and count the stained reticulocytes under the microscope. There also are alternative procedures, either manual or automatic, which employ fluorescent dyes such as acrydine orange, pyronine Y, thioflavin T, auramine O, thiazole orange and so on.
The ultravital staining technique using new methylene blue has been employed as the standard method for enumerating reticulocytes but being a manual procedure, this technique is time-consuming and complicated and has the drawback of individual difference according to different observers.
The determination technique employing acrydine orange is either manual or automatic as disclosed in, inter alia, U.S. Pat. Nos. 4,336,029, 4,325,706 and 4,284,412. Thus, acrydine orange couples itself to the RNA component of reticulocytes to produce a red fluorescence and the number of reticulocytes is determined by measuring the emissions. However, the RNA is precipitated by acrydine orange and localized within the cell so that the quantitation of fluorescence tends to lack accuracy. Moreover, this dye has a high affinity for the quartz cell and plastic materials constituting the circuitry of a flow cytometer.
Determination with pyronine Y requires a prior formalin fixation of red blood cells and is disadvantageous in that the technique is complicated and time-consuming. Furthermore, the fluorescent quantum efficiency of pyronine Y is so low that no sufficient sensitivity can be expected.
Thioflavin T is disclosed in Japanese Patent Application Kokai S-59-142465 as a reticulocyte stain but since it requires sufficient washing for reducing the noise of nonspecific fluorescence, determination with this dye has the disadvantage that it requires a complicated and time-consuming procedure.
Auramine O and thiazole orange produce fluorescent emissions when excited by blue light but when a helium-cadmium laser is used as a light source in flow cytometry, the required equipment has to be large in size and expensive and the RNA specificity of these dyes is relatively lower than their specificity to DNA and protein.
Thus, the prior art determination techniques for enumerating reticulocytes using a flow cytometer have their own problems relating to sensitivity, accuracy and/or cost.
It is, therefore, a primary object of this invention to provide a means which enables sensitive, accurate and inexpensive determinations of reticulocytes using a dye suitable for staining reticulocytes.